Only recently has attention been paid to the initiation of responses of human polymorphonuclear leukocytes (PMN) and to biochemical and physiological events which take place during the first minute of granulocyte stimulation. Measurements of these earliest events and determinations of the order in which they occur allows a separation of causes from effects. By the use of techniques which provide a time resolution measured in seconds (rather than minutes), we have established a temporal order for membrane hyperpolarization, cAMP changes, lysosomal enzyme release, and superoxide anion generation. Our objectives are to place these and the following studies into an explanatory framework which would permit a logical description of how surface ligand interactions are translated into cellular responses: 1) What is the role of transient elevations in cellular cAMP which accompany surface stimulation of PMN? 2) Whether protein carboxymethylation is a critical step in PMN responses? 3) Whether calmodulin is critical in PMN responses? 4) To what extent are rapid changes in membrane-bound calcium and magnesium important during stimulation? 5) Do the rapid changes in fluorescence of l-anilino-8-naphthalene sulfonate which accompany stimulation reveal a critical change in membrane structure? 6) Does stimulation of PMN produce rapid changes in membrane microviscosity (examined by means of fluorescence deplorization and pyrene eximer formation)? These studies will use PMN isolated from venous blood of informed volunteers. Such investigations, in combination with ion flux studies currently being conducted in this laboratory, should help elucidate the roles of these events in the sequence of stimulus-secretion coupling.